Purified cell populations have many applications in biomedical research and clinical therapies (Auditore-Hargreaves et al., Bioconjug. Chem. 5:287-300, 1994; and Weissman, Science 287:1442-1446, 2000). Often, cells can be separated from each other through differences in size, density, or charge. However, for cells of similar physical properties, separation is often accomplished by exploiting differences in the presentation of molecules on the cell surface. Cell-affinity chromatography is based on this approach, most often by employing immobilized antibodies to specific cell surface antigens. Such affinity column separations require several distinct steps including incubation of the cells with the antibody, elution of the cells, cell collection, and release of the conjugated antibody, with each step reducing the overall yield of cells and increasing the cost of the process.
There exists a need for obtaining cellular samples from donors that are enriched in desired biological targets. Because a heterogeneous sample may contain a negligible amount of a biological entity of interest, the limits of separation methods to provide viable and potent biological target in sufficient purity and amount for research, diagnostic or therapeutic use are often exceeded. Because of the low yield after separation and purification, some cell-types, such as stem cells, progenitor cells, and immune cells (particularly T-cells) must be placed in long-term culture systems under conditions that enable cell viability and clinical potency to be maintained and under which cells can propagate (cell expansion). Such conditions are not always known to exist. In order to obtain a sufficient amount of a biological target, a large amount of sample, such as peripheral blood, must be obtained from a donor at one time, or samples must be withdrawn multiple times from a donor and then subjected to one or more lengthy, expensive, and often low-yield separation procedures to obtain a useful preparation of the biological target. Taken together, these problems place significant burdens on donors, separation methods, technicians, clinicians, and patients. These burdens significantly add to the time and costs required to isolate the desired cells.
Stem cells are capable of both indefinite proliferation and differentiation into specialized cells that serve as a continuous source for new cells that comprise such tissues as blood, myocardium and liver. Hematopoietic stem cells are rare, pluripotent cells, having the capacity to give rise to all lineages of blood cells (Kerr, Hematol./Oncol. Clin. N. Am. 12:503-519, 1998). Stem cells undergo a transformation into progenitor cells, which are the precursors of several different blood cell types, including erythroblasts, myeloblasts, monocytes, and macrophages. Stem cells have a wide range of potential applications, particularly in the autologous treatment of cancer patients.
Typically, stem cell products (true stem cells, progenitor cells, and CD34+ cells) are harvested from the bone marrow of a donor in a procedure, which may be painful, and requires hospitalization and general anesthesia (Recktenwald et al., Cell Separation Methods and Applications, Marcel Dekker, New York, 1998). More recently, methods have been developed enabling stem cells and committed progenitor cells to be obtained from donated peripheral blood or peripheral blood collected during a surgical procedure.
Progenitor cells, whether derived from bone marrow or peripheral blood, can be used to enhance the healing of damaged tissues (such as myocardium damaged by myocardial infarction) as well as to enhance hematologic recovery following an immunosuppressive procedure (such as chemotherapy). Thus, improved approaches to purify stem cells ex vivo, or to “re-address” circulating stem cells in vivo, has great potential to benefit the public health.
Hematopoietic stem and precursor cells (HSPC) are able to restore the host immune response through bone marrow transplantation, yet the demand for these cells far exceeds the available supply. HSPC also show great promise for treatment of other hematological disorders. HSPC are believed to adhesively roll on selectins during homing to the bone marrow in a manner analogous to the (much better understood) process of leukocyte trafficking Previous work has demonstrated that CD34+ cells (showing a marker of stem cell immaturity) roll more slowly and in greater numbers than more differentiated CD34− cells. By exploiting this difference in rolling affinity it is possible to construct a flow chamber device for continuous separation and purification of CD34+ cells from an initial mixture of blood cells, while maintaining viability of the cells for subsequent use in clinical applications. Such a process holds several distinct advantages over current affinity column methods. The feasibility of cell separation based on rolling affinity has been demonstrated by others for artificial adhesive microbeads, and by us for live stem cell populations.
CD34 is a surface marker of stem cell immaturity. Recent work has shown that CD34+ cells from the adult bone marrow and fetal liver roll more slowly and to a greater extent on P- and L-selectin, compared to CD34− cells (Greenberg et al., Biophys. J. 79:2391-2403, 2000). Further, Greenberg et al. (Biotechnol. Bioeng. 73:111-124, 2001) demonstrated that rolling affinity-based separations of carbohydrate-coated microspheres is possible. However, there remains a need for methods and apparatus for separation of a particular type of cells, particularly, immature stem cells from other cells, such as more mature cells, in a continuous, single-pass, high-throughput flow chamber.